WalK(S221P) Mutation Promotes the Production of Staphylococcus aureus Capsules Through an MgrA-Dependent Pathway

Abstract

Staphylococcus aureus is a vital pathogen causing clinical infections. Capsules are important virulence factors for S. aureus. This study investigates the regulatory mechanisms underlying capsule production in S. aureus. Bacterial strains XN108 and Newman were used, and combined approaches like RNA sequencing (RNA-seq), RT-qPCR, transmission electron microscopy (TEM), gene reporter, and electrophoretic mobility shift assay (EMSA) were performed to test the role and mechanism of WalK(S221P) mutation in S. aureus capsule production. RNA-seq showed an increased expression of cap genes in the WalK(S221P)-carried S. aureus XN108 relative to the mutation-cured XN108-R. TEM and capsular polysaccharide determination demonstrated that XN108 produced more capsules than XN108-R did. Similar results were presented in the WalK(S221P)-contained K-Newman versus the wild-type Newman. RT-qPCR screening showed an increasing expression of the mgrA gene in XN108 versus XN108-R. Gene reporter and EMSA analysis revealed that WalK(S221P) mutation promoted S. aureus capsule production through MgrA. Deletion of mgrA decreased the WalK(S221P)-mediated capsule yield. Moreover, WalK(S221P) mutation remarkably increased the tolerance of S. aureus to whole blood killing and microphage phagocytosis. Overall, these data provide mechanistic insights into the effect of WalK(S221P) on the capsule production of S. aureus, which may set down foundations for future S. aureus virulence investigations.

1. Introduction

Staphylococcus aureus (S. aureus) is an important pathogen that can cause a variety of diseases, including skin/soft tissue disorders, pneumonia, myocarditis, and sepsis [1]. S. aureus often results in the high mortality of patients suffering with influenza or SARS-CoV-2 infections [2,3]. According to a report from the World Health Organization, there are approximately 20,000 S. aureus bacteremia-related deaths in the United States each year [4]. In China, S. aureus is ranked as the most clinically important Gram-positive bacteria according to the China Antimicrobial Surveillance Network (http://www.chinets.com, accessed on 23 December 2024). Virulence factors are morbigenous weapons used by pathogenic bacteria to cause diseases [5]. The notorious S. aureus can secrete plenty of virulence factors, such as enterotoxins, hemolysins, exfoliative toxins, leucocidins, proteases, and toxic shock syndrome toxin 1 (TSST-1) [6,7,8]. Capsules are constituted with diverse capsular polysaccharides (CPs) and also important virulence factors for S. aureus [9]. S. aureus capsules can protect bacterial cells from phagocytosis and facilitate bacteria to escape host immune inactivation [10]. To date, 11 serotypes of S. aureus CPs have been categorized [11]. However, more than 90% of clinically isolated S. aureus strains produce type 5 (CP5) or type 8 CPs (CP8) [12,13].

The production of S. aureus capsules is controlled by a cap operon, which has 16 genes (capA–capP) encoding various enzymes involved in CP biosynthesis and transportation [14]. The expression of the cap gene cluster is strictly controlled during S. aureus growth, showing a repression before the exponential phase, a priming expression in the late exponential stage, and a maximum production at the stationary phase [15]. This timely expression profile of S. aureus cap genes may be ascribed to a regulatory network that governs the cap promoter activity. Recently, Keinhörster et al. have revisited the regulation of the cap gene cluster in S. aureus and found that the promoter element of cap operon (Pcap) contains a major sigma B (SigB)-dependent promoter (PsigB) and a weak SigA-dependent promoter (PsigA) [16]. An array of positive regulators such as MgrA, SpoVG, and RbsR and negative modulators like Rot, CodY, and SaeSR control the expression of the cap gene cluster by directly affecting PsigB or PsigA activities [16,17,18,19]. MgrA is a small transcriptional modulator that affects the expression of a large number of genes [20,21]. Lei and Lee reported that MgrA directly binds to the Pcap and affects the SigA-dependent promoter in S. aureus [19]. Other regulators such as SigB, SbsDC, and the two-component systems (TCS) AgrCA and ArlSR can influence CP biosynthesis through MgrA [20,22,23,24]. WalKR is the only essential TCS for S. aureus survival by regulating bacterial cell wall metabolism [25]. The expression of approximately 200 genes in S. aureus is directly and indirectly regulated by WalKR [26]. However, whether WalKR affects CP production in S. aureus has not been determined.

In the previous studies, we characterized a vancomycin-intermediate S. aureus (VISA) strain XN108 and found that a TCS element mutation, WalK(S221P), is associated with the vancomycin resistance and virulence of XN108 [26,27,28,29,30]. The present study aims to investigate the role and mechanism of WalK(S221P) mutations in S. aureus capsule production. RNA sequencing (RNA-seq) and RT-qPCR analysis showed that the WalK(S221P) mutation resulted in the upregulation of S. aureus cap genes. Transmission electron microscopy (TEM) and CP quantitative determination showed that the WalK(S221P) mutation-carried S. aureus strains XN108 and K-Newman produced more CPs than their counterparts. Further, the LacZ gene reporter, the electrophoretic mobility shift assay (EMSA), and CP observation and quantitation revealed that WalK(S221P) mutation promoted capsule production through an MgrA-dependent pathway. The inactivation of mgrA impaired the WalK(S221P)-mediated CP yield and, when complemented with mgrA, restored the CP biosynthesis. Moreover, WalK(S221P) mutation increased the survival of S. aureus in the whole-blood inactivation and after phagocytosis by microphages. Overall, our data presented a WalKR-MgrA axis that controls the capsule production in S. aureus, and the WalK(S221P) mutation may enhance the CP yield through the upregulation of mgrA expression.

2. Materials and Methods

2.1. Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. The methicillin-resistant S. aureus (MRSA) and VISA strain XN108 were isolated from a burn patient suffering from an acute S. aureus infection [27]. The methicillin-susceptible S. aureus (MSSA) strain Newman (NCTC 8178) was provided by Prof. Lu Yu (Jilin University). XN108-R and K-Newman were constructed as described previously [26]. A pLI50 plasmid was used for gene complementation, plasmid pOS1 was used to construct reporter, pET-28a was used to prepare recombinant proteins, and Escherichia coli-S. aureus shuttle vector pBT2 was used for gene deletion. S. aureus and its derivative strains were cultured in a brain heart infusion (BHI, Oxoid, Hampshire, UK), and chloramphenicol (20 μg/mL) was added when necessary. E. coli strains were grown in a Luria–Bertani (LB) medium with ampicillin (100 μg/mL) or kanamycin (50 μg/mL) as needed.

2.2. RNA-Seq

S. aureus cells XN108 or XN108-R at the logarithmic phase were harvested, and the total RNA was prepared with an RNAprep Pure Cell/Bacteria Kit (TianGen, Beijing, China). Three biological replicate samples were used for RNA-seq, and the cDNA libraries were generated from each RNA sample with a TruSeq Illumina Kit (San Diego, CA, USA) as described [29]. RNA-seq was performed in Novogene Co., Ltd. (Beijing, China) with the Illumina HiSeqTM2500 sequencing system. The analysis of the results was performed according to the protocol recommended by the manufacturer (Illumina Inc., San Diego, CA, USA). The software DESeq version 1.10.1 was used to analyze differentially expressed genes (DEGs), as previously described [26]. The RNA-seq raw data were submitted and deposited in the GEO DataSets with an ID code of GSE127706.

2.3. RT-qPCR

S. aureus strains XN108 and XN108-R were cultured in a BHI at 37 °C for 12 h. Total RNA from each strain were extracted with the RNAprep Pure Cell/Bacteria Kit (TianGen, China). The relevant cDNA samples were obtained using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Afterward, the quantitative polymerase chain reaction (qPCR) was conducted to detect the relative expression level of each target gene to the reference gyrB, as previously described [26]. The primers used in this study were provided in Table S1.

2.4. CP Extraction and Quantitation

The CP extraction was performed as previously described with some modifications [24]. Briefly, the S. aureus strains were cultured in a BHI at 37 °C for 2 d. Bacterial cells were harvested by centrifugation at 8000× g for 10 min, resuspended in phosphate-buffered saline (PBS, pH 7.2), autoclaved at 121 °C for 50 min, and then centrifuged at 8000× g for 10 min. The supernatant was filtered through a 0.22 μm membrane (Millipore, Burlington, MA, USA) and kept at 4 °C overnight. Precooled ethanol (4 °C) was added to a final concentration of 80% (v/v) and incubated overnight at 4 °C to precipitate the CPs. After washing twice with precooled 80% (v/v) and once with 96% (v/v) ethanol, the CPs were collected by centrifugation at 15,000× g for 10 min, dissolved in PBS, dialyzed in ddH₂O for 2 d, and finally lyophilized. Then, the lyophilized sample was dissolved in buffer A (2 mM MgSO₄, 50 mM Tris-HCl, pH 7.5) to a concentration of 10 mg/mL and treated with DNase I (Solarbio, Beijing, China) and RNase A (Sigma, St. Louis, MO, USA) at 1 mg/mL at 37 °C for 6 h. Subsequently, the mixture was treated with 2 mg/mL Protein K (Merck Millipore, Darmstadt, Germany) at 65 °C for 6 h. After centrifugation at 15,000× g for 15 min, the supernatant was ultrafiltered using a 30 kDa MWCO centrifugal tube (Merck Millipore) and lyophilized. The lyophilized material was dissolved in PBS to a concentration of 20 mg/mL, and 0.05 M NaIO₄ was added to remove teichoic acids. The resulting mixture was kept in dark conditions until the value of optical density at 233 nm (OD₂₃₃) was stable. The reaction was stopped by adding 10% (v/v) 16 M ethylene glycol. Ultrafiltration with a 100 kDa MWCO centrifugal tube was performed to collect the filtered solution, which was further ultrafiltered with a 50 kDa MWCO centrifuge tube to obtain the CP solution. After dialysis in ddH₂O for 2 d, the CP sample was lyophilized and kept at −80 °C.

2.5. TEM Observation

To analyze the morphology of the capsule structure, S. aureus strains of interest were cultured on Columbia agar plates supplemented with 2% (m/v) NaCl (Thermo Scientific, Waltham, MA, USA) at 37 °C for 24 h. Bacterial cells were collected and washed with PBS. Then, the cell sample was prepared with a method of lysine acetate-based formaldehyde/glutaraldehyde ruthenium red osmium fixation procedure, as previously described [32]. In brief, S. aureus cells were fixed with 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde in a cacodylate buffer supplemented with 0.075% (m/v) ruthenium red and 0.075 M lysine acetate on ice for 20 min. After washing with the cacodylate buffer containing ruthenium red, bacteria were secondly fixed with formaldehyde and glutaraldehyde for 3 h, followed by fixation with 1% (m/v) osmium in the cacodylate buffer with ruthenium red for 1 h at room temperature. Washing was performed five times with the ruthenium red-carrying cacodylate buffer, and bacterial samples were then dehydrated with gradient concentrations of ethanol (10, 30, 50, 70, 90, and 100%) on ice for 30 min per step. After infiltration with the acrylic resin (London Resin Company, London, UK), the ultrathin sections were prepared. Finally, bacterial sample sections were observed under a TECNAI 10 transmission electron microscope (Philips, The Netherlands) and photographed.

2.6. β-Galactosidase Assay

The DNA fragments carrying the mgrA-promoter (PmgrA) and cap-promoter (Pcap) were obtained by PCR using primer pairs pOS-mgrA-F/R and pOS-cap-F/R (Table S1), respectively. Subsequently, PCR products were digested with BamH I and EcoR I and cloned into pOS1 plasmid to generate pOS-mgrA and pOS-cap. The resulting plasmids were transformed into S. aureus RN4220 for restriction modification, followed by transformation into XN108, XN108-R, and XN108ΔmgrA.

The β-galactosidase assay was conduct as described [20]. In brief, S. aureus strains transformed with pOS-mgrA or pOS-cap were cultured in a BHI medium supplemented with chloramphenicol (10 μg/mL) at 37 °C overnight. On the next day, the culture was inoculated in a fresh BHI broth (1:200 dilution) and cultivated at 37 °C for 8 h. Bacterial cells were harvested by centrifugation at 10,000× g for 1 min and resuspended in an AB buffer (100 mM NaCl, 100 mM KH₂PO₄, pH 7.0) with consistent OD₆₀₀ values. For each reporter strain, 10 μL of the bacterial solution was added into 85 μL of the AB buffer, and lysostaphin (Sigma, St. Louis, MO, USA) at a final concentration of 5 μg/mL was added to the lyse bacterial cells by incubating at 37 °C for 2 h. Then, 10 μL of the mixture was transferred into a well of 96-well plates with 15 μL of 1% (v/v) Triton X-100. Subsequently, 30 μL of 4-methylumbelliferyl-β-d-galactoside (Sigma, St. Louis, MO, USA) was added at a final concentration of 1 mg/mL, and the plate was incubated at 37 °C for 1 h. Finally, the reaction was stopped by adding 75 μL of a stop buffer (15 mM EDTA, 300 mM glycine, pH 11.2). The fluorescence intensity was measured using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

2.7. Protein Expression and Purification

The recombinant proteins WalK and WalR were prepared as described [16]. To obtain recombinant MgrA, its gene was amplified by PCR with primers PET28a-mgrA-F/R (Table S1) from the genomic DNA of S. aureus XN108. A 6 × His tag was introduced by primer PET28a-mgrA-R and fused with the C-terminal of MgrA protein. The PCR product was digested with BamH I and SaI I and cloned into a pET28a vector to generate pET-mgrA. Subsequently, the vector was transformed into E. coli strain BL21(DE3), and 0.1 μM isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of MgrA. The MgrA proteins were purified as previously described [16]. The purified protein was quantified using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) and stored at −80 °C until use.

2.8. EMSA

The biotin-labelled DNA probes of the cap (Pcap), mgrA (PmgrA), and agr (agr) gene promoters were obtained from S. aureus XN108 genomic DNA by PCR with the primers cap-F-biotin/cap-R, mgrA-F-biotin/mgrA-R, and agr-F-biotin/agr-R, respectively (Table S1). An unrelated DNA fragment hu was also amplified by PCR using the primer pair hu-F/hu-R to serve as a nonspecific control. The unlabeled DNA fragment amplified with the primer pair mgrA-F/mgrA-R or cap-F/cap-R was used in the binding competition. The EMSA was performed as previously described [16]. Briefly, 0.25 nM of Pcap or PmgrA probes was added to various amount of WalR (0 to 8 μM) activated with 1 μM of WalK and 5 mM of ATP in 20 μL of a reaction buffer containing 1 mM EDTA, 100 mM NaCl, and 50 mM Tris−HCl at a pH of 8.0 [23]. For MgrA binding to Pcap, 0.5 nM of Pcap DNA was added to diverse concentrations of MgrA (0 to 4 μM) in 20 μL of the reaction buffer containing 20 mM HEPES (pH 8.0), 1 mM DTT, 100 mM NaCl, 1% (v/v) glycerol, and sheared salmon sperm DNA (3 ng/μL), as described [20]. After incubation at 25 °C for 30 min, the reaction mixture was separated on 6% (m/v) natural acrylamide gels and electrophoresed with a 0.5 × TBE buffer at 100 V for 120 min. Subsequently, the samples were electrotransferred onto a positively charged nylon membrane (Beyotime, Shanghai, China), cross-linked with UV, and developed with a Chemiluminescent Nucleic Acid Detection Kit (Thermo scientific). The image was obtained using a Chemiluminescence imager (VILBER, Collégien, France).

2.9. Genetic Construction

The gene deletion and complementary S. aureus strains were constructed as previously described [26]. Taking the generation of XN108ΔmgrA as an example, about 1000 bp DNA fragments flanking left and right DNA sequences of the mgrA gene were amplified from XN108 genomic DNA by PCR with the primer pairs pBT2-mgrA-left-F/mgrA-left-R, and mgrA-right-F/pBT2-mgrA-right-R (Table S1), respectively. The left and right DNA fragments were fused by over-lap PCR and cloned into pBT2 to obtain pBT2ΔmgrA. After modification in S. aureus RN4220, the pBT2ΔmgrA was electroporated into the S. aureus strain XN108. The seamless mgrA deletion mutant XN108ΔmgrA was selected via homologous recombination and verified by PCR and DNA sequencing.

For mgrA complementation, the mgrA gene fragment with its promoter region was amplified by PCR using primers pLI/mgrA-F/R (Table S1) and cloned into the shuttle plasmid pLI50 to achieve pLI-mgrA. After modification, the plasmid was electroporated into XN108ΔmgrA to obtain a XN108ΔmgrA/pLI-mgrA complemented strain.

2.10. Bacterial Growth Curve

The growth curves of S. aureus XN108 and its derivatives were detected as described [33]. Briefly, bacteria were cultured overnight at 37 °C in a BHI medium with shaking, and the 200 μL overnight culture was inoculated into 20 mL of the fresh BHI broth (1:100 dilution) in a sterile flask (50 mL). The OD₆₀₀ values were determined every hour for 24 h, and the growth curve was drawn by using OD₆₀₀ values throughout the culture time.

2.11. Whole Blood Killing

Overnight cultures of S. aureus strains were diluted 1:100 into the fresh BHI and grown at 37 °C for 6 h. Bacterial cells were collected by centrifugation, washed twice, and diluted in PBS to adjust to an OD600 of 0.5 (about 10⁸ CFU/mL⁻¹). Approximately 10⁶ colony forming unit (CFU) cells in 100 µL PBS were mixed with 900 µL of fresh Sprague–Dawley mouse blood (SCBS, Shanghai, China). The tubes were incubated at 37 °C with slow rotation. Approximately 200 µL mixture was taken after 1 or 2 h of incubation, and 0.5% (m/v) saponin was used to lyse blood cells. The samples were then serially diluted for bacterial counting with BHI agar plates. The survival rate (%) was calculated as (CFU_{time point}/CFU_{initial input}) × 100.

2.12. Bacterial Survival After Macrophage Phagocytosis

RAW264.7 macrophages were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum at 37 °C with 5% CO₂. S. aureus cells at the exponential growth phase were collected and resuspended in DMEM. RAW264.7 cells were infected with the S. aureus strain at a multiplicity of infection (MOI) of 10 in 24-well plates. At 1 h post infection (hpi), the supernatant was removed, and the macrophages were washed with sterile PBS. About 1 mL DMEM containing lysostaphin (1 mg/mL) and gentamicin (50 µg/mL) was added to eliminate extracellular bacteria by incubating at 37 °C for 1 h. Macrophages were then cultured for 4 or 24 h at 37 °C with 5% CO₂. After lysis with PBS containing 1% Triton X-100, the bacterial CFUs after phagocytosis were counted with BHI agar plates. The experiment was repeated three times.

2.13. Statistical Analysis

The experimental data were analyzed using GraphPad Prism 9.3.1. Data from two independent groups were treated with unpaired two-tailed student’s t-tests, and a p value less than 0.05 was considered statistically significant.

3. Results

3.1. WalK(S221P) Mutation Enhances Capsule Production of S. aureus

RNA-seq has been performed to explore the role of WalK(S221P) mutation, and the results showed that the expression levels of all 16 genes involved in the CP biosynthesis of S. aureus were remarkably upregulated in XN108 compared with those in the WalK(S221P)-reverted strain XN108-R (Figure 1A). This phenomenon was further confirmed by the RT-qPCR detection of capA, capG, and capL gene expression levels in XN108 versus XN108-R, and a similar expression profile was revealed (Figure 1B). We next detected whether the upregulated cap genes after WalK(S221P) mutation can increase capsular production in S. aureus. The CPs of XN108 and XN108-R were extracted and quantified with a phenol–sulfuric acid method as described [34,35]. As shown in Figure 1C, S. aureus XN108 produced more CPs [(116.1 ± 3.8) µg/g cell weight] than XN108-R [(103.2 ± 1.7) µg/g] did (p < 0.01). Moreover, TEM revealed a significantly thicker capsule of XN108 [(39.3 ± 16.7) nm] in comparison to XN108-R [(19.5 ± 8.2) nm, p < 0.001, Figure 1D,E]. These results demonstrated that the WalK(S221P) mutation has a positive regulatory effect on the capsule synthesis of the S. aureus strain XN108.

Most clinical S. aureus isolates carry a CP5 or CP8 capsule [36]. The S. aureus strain Newman (CP5) was used to detect whether the observed role of WalK(S221P) mutation in the capsular production of XN108 (CP8) occurs in CP5 isolates. An allelic replacement strain was generated by substituting walK in the Newman with that from XN108 (walk(S221P)), namely K-Newman [26]. RT-qPCR detection exhibited that the expressions of the capA, capG, and capL genes were considerably increased in K-Newman compared with those in the wild-type Newman (Figure S1A). Accompanying the increased cap gene expression, K-Newman presented a thickened capsule relative to the wild-type Newman (Figure S1B,C). Overall, these results indicated that WalK(S221P) mutation can promote the production of S. aureus capsules.

3.2. WalKR Controls Cap Expression Indirectly

To determine whether WalKR controls the expression of cap genes, a reporter carrying lacZ gene controlled by the cap promoter (pOS-cap) was constructed, and a β-galactosidase assay was performed after the transformation of pOS-cap into S. aureus strains XN108 and XN108-R. The results showed that β-galactosidase activity in XN108 was significantly increased compared with that in the XN108-R (p < 0.05, Figure 2A), suggesting a positive effect of WalK(S221P) mutation on the promoter activity of cap gene cluster. The response regulator WalR executes its role by binding a consensus motif (5′-TGTWAH-N5-TGTWAH-3′) located upstream of the target genes in S. aureus [25]. However, no predicted consensus motif of WalR was identified in the promoter regions of cap operon in S. aureus XN108. To further verify the direct regulatory function of WalR on cap operon, the His-tagged recombinant proteins WalK and WalR were prepared (Figure 2B). The EMSA revealed that the WalK-phosphorylated WalR did not bind the biotin-labelled cap gene promoter probe but bound to the biotin-labelled agr gene promoter probe, which served as a positive control (Figure 2C). Collectively, these data showed that WalKR may indirectly control the expression of cap genes.

3.3. WalK(S221P) Upregulates Cap Gene Expression Through MgrA

To detect the molecule that mediates the effect of WalK(S221P) mutation on cap gene expression, RT-qPCR was performed to determine the altered regulatory genes in S. aureus with WalK(S221P) mutation. The results showed that the expression of mgrA was mostly increased in XN108 compared with that in XN108-R, followed by spoVG, whereas other detected regulators did not change their expression levels in XN108 versus XN108-R (Figure 3A). To further detect whether WalK(S221P) mutation affects mgrA expression, a reporter plasmid (pOS-mgrA) with the mgrA gene promoter-lacZ fusion was constructed. After transformation into XN108 and XN108-R, LacZ activity analysis revealed that WalK(S221P)-carrying XN108 exhibited remarkably stronger mgrA promoter activity than XN108-R did (Figure 3B). The EMSA showed that WalK-phosphorylated WalR could specifically bound to the mgrA promoter DNA probe (Biotin PmgrA) in a dose-dependent manner (Figure 3C). The binding was outcompeted by the 300-fold addition of unlabelled PmgrA probe DNA; however, the 300-fold addition of an unrelated hu DNA fragment did not inhibit the binding. These data demonstrated that WalKR can directly control mgrA expression, and WalK(S221P) mutation resulted in the upregulation of mgrA in XN108.

MgrA is reportedly used to regulate cap gene expression [19]. Thus, a mutant with mgrA deletion was constructed (Figure S2). S. aureus strains XN108 and XN108ΔmgrA were, respectively, transformed with pOS-cap plasmid to test the effect of MgrA on cap expression. As shown in Figure 3D, the LacZ assay revealed that the activity of the cap promoter in XN108 was stronger than that in the XN108ΔmgrA, indicating a positive role of MgrA on cap gene expression. Next, the recombinant MgrA protein was prepared (Figure 3E), and the EMSA showed that MgrA could bind to the cap gene promoter DNA in a dose-dependent manner (Figure 3F). The binding ability of MgrA was outcompeted by the 300-fold addition of unlabeled DNA fragments of PmgrA, but not the irrelevant DNA fragment hu (Figure 3F). Collectively, these results showed that MgrA directly controls cap gene expression, and WalK(S221P) mutation promoted capsule production in S. aureus through MgrA.

3.4. Deletion of mgrA Reduces Capsule Production

To determine whether the MgrA-mediated effect of WalK(S221P) mutation on cap gene expression influences S. aureus capsule production, a complementary strain was constructed by the transformation of a pLImgrA expression plasmid into XN108ΔmgrA. The deletion and complementation of mgrA did not affect the growth of the bacteria XN108ΔmgrA and XN108ΔmgrA/pLImgrA (Figure 4A). TEM observed that the capsular thickness of XN108ΔmgrA [(10.0 ± 7.7) nm] substantially decreased compared with that of the wild-type strain [(39.3 ± 16.7) nm], and complementation with mgrA partially increased the capsular thickness of XN108ΔmgrA/pLImgrA [(18.3 ± 7.8) nm, Figure 4B,C]. The quantification analysis revealed significantly reduced CPs in XN108ΔmgrA [(24.6 ± 1.5) µg/g] and elevated capsular production in XN108ΔmgrA/pLImgrA [(107.9 ± 8.6) µg/g], comparable to that of the wild-type XN108 [(116.1 ± 3.8) µg/g, Figure 4D]. These results showed that mgrA plays an important role in the promotion of capsule biosynthesis in S. aureus with WalK(S221P) mutation.

3.5. WalK(S221P)- and MgrA-Mediated Capsule Production Promotes Bacterial Intracellular Survival

S. aureus capsules can protect bacteria from phagocytosis, thereby enhancing bacterial intracellular survival [37,38,39]. Neutrophiles that circulate in the blood can inactivate bacteria by oxidative killing [40]. Therefore, whole-blood killing was performed to determine if the enhanced capsule contributes to S. aureus survival. The results exhibited that the survival rate of XN108-R and XN108ΔmgrA after 1 or 2 h incubation with Sprague–Dawley mouse blood considerably decreased compared with that of XN108 (Figure 5A). Macrophages are the first line of host defence against invading bacteria [41]. To detect whether WalK(S221P)-promoted capsule production contributes to S. aureus survival within macrophages, we assessed the survival ability of XN108 or its derivatives within RAW264.7 macrophages. As shown in Figure 5B, XN108-R and XN108ΔmgrA remarkably reduced their survival numbers within RAW264.7 macrophages compared with the wild-type XN108 after 24 h of culture. However, comparable survivals of XN108-R, XN108ΔmgrA, and XN108 were revealed after 4 h of culture within RAW264.7 macrophages. Overall, these data demonstrated that WalK(S221P) mutation and MgrA promoted the tolerance of S. aureus cells to whole blood- and macrophage-mediated killing by upregulating the production of bacterial capsules.

4. Discussion

S. aureus is a versatile pathogen equipped with an array of virulence factors, which facilitate bacterial colonization, infection, and immune evasion [7,8]. As one of the important immune escape molecules, the capsule plays a vital role in S. aureus infections [12]. The regulation of capsule production in S. aureus is complex, and many regulators can affect the expression of the cap gene cluster directly or indirectly [17,18,19,22,24,42]. WalKR is an essential TCS of S. aureus and is responsible for the regulation of cell wall metabolism, protein biosynthesis, nucleotide metabolism, and DNA replicative initiation [25,43]. The previous studies have demonstrated that WalK(S221P) mutation contributed to VISA phenotypes, such as increased vancomycin resistance and attenuated bacterial virulence [22,26]. However, the effects of WalKR mutations on S. aureus capsule production are unclear. In the present study, we demonstrated that the WalK(S221P) mutation promoted the capsule production of S. aureus by upregulating mgrA expression. Consistently, the isogenic deletion of mgrA decreased CP biosynthesis, indicating crucial roles of WalK(S221P) and MgrA in modulating the immune escape system in S. aureus.

Capsule production is determined by the expression of the cap gene cluster, which encodes the required enzymes for CP biosynthesis [14]. RNA-seq analysis and RT-qPCR detection exhibited that WalK(S221P) mutation resulted in upregulated cap gene expression in the CP8 strain XN108 compared with the WalK(S221P)-reverted counterpart XN108-R. This phenomenon was verified in the CP5 strain K-Newman versus wild-type Newman. WalKR comprises a sensor histidine kinase WalK and a DNA-binding response regulator WalR [44]. Upon activation by certain signals, the H385 of WalK is auto-phosphorylated, and the phosphoryl group is then transferred to the aspartate residue 53 (D53) of WalR, which functions as a transcriptional regulator [25]. Therefore, we generated a LacZ reporter, and the β-galactosidase assay revealed a positive effect of WalK(S221P) mutation on the promoter activity of the cap gene cluster, whereas the EMSA showed no direct binding of WalK-activated WalR to the cap promoter DNA probe, indicating an indirect role of WalKR in cap gene expression.

RT-qPCR was performed to screen the potential molecules in mediating the WalKR regulation of cap gene cluster. The results demonstrated the expression of mgrA was mostly upregulated in the WalK(S221P) mutation-carrying strain. MgrA is a member of the MarR (multiple antibiotic resistance regulator) protein family, which can regulate the expression of virulence factors, including alpha-toxin, coagulase, protein A, nuclease, extracellular serine proteases, and capsule [21]. Luong et al. reported that the ArlRS TCS modulated S. aureus capsule production through MgrA [20]. Crosby et al. demonstrated that phosphorylated ArlR bound to the promoter regions to activate mgrA expression [45]. We showed that WalK-activated WalR could bind to the mgrA promoter DNA probe and MgrA could directly bind to the Pcap probe. Thus, WalKR and MgrA orchestrated a WalKR-MgrA axis to control the capsule production of S. aureus. Structurally, WalK(S221P) mutation occurs in the HAMP (histidine kinases, Adenylylcyclases, Methyl binding proteins, and Phosphatases) domain of WalK, which connects the extracellular sensory and intracellular signalling domains (Figure 6). In a previous study, the in vitro phosphorylation assay demonstrated that WalK(S221P) mutation presented decreased autophosphorylation, which affected the activation of WalR [28]. WalKR may negatively control mgrA expression, and the WalK(S221P) mutation reduced the effect of WalKR on the mgrA promoter, thereby upregulating mgrA to enhance CP biosynthesis in S. aureus. However, the fine regulatory mechanism of the WalKR-MgrA axis requires further investigation, which will guide our next work.

As an immune escape molecule, WalK(S221P)-mediated capsule production is supposed to enhance bacterial survival [7,46]. In this study, the whole blood killing and macrophage phagocytosis confirmed this conclusion. The survival rate of XN108-R after incubation with mouse whole blood for 1 or 2 h substantially reduced compared with that of XN108, and a similar result was revealed in the case of XN108ΔmgrA. Consistently, the survival numbers of XN108-R and XN108ΔmgrA remarkably decreased after 24 h of phagocytosis by RAW264.7 macrophages when compared with that of XN108. These results further confirmed that MgrA mediated the promotion of WalK(S221P) mutation to S. aureus capsule production. Therefore, enhancing WalKR activity and MgrA inhibition could be promising strategies to inhibit CP production and facilitate the clinical control of S. aureus infections.

5. Conclusions

We demonstrated the role of WalK(S221P) mutation in promoting the capsule production of S. aureus. Moreover, WalK(S221P) increased CP biosynthesis through MgrA, a member of the MarR protein family, which is a first-line regulator governing cap gene expression (Figure 6). In addition, WalK(S221P)-mediated capsule yield contributed to S. aureus survival. Our study provided a WalKR-MgrA axis in controlling CP biosynthesis in S. aureus, which may establish a connection between bacterial virulence and strain metabolism. However, the present study has several limitations. First, only two strains (one VISA strain XN108 and an MSSA strain Newman) were tested, and more strains are needed to determine the role of WalK(S221P)-MgrA in promoting capsule production in S. aureus. Second, WalKR may have various mutations except for WalK(S221P), and other mutations could play similar roles. More mutations like WalK(G223D), WalK(V381I), and WalR(K208R) are needed to further confirm the roles of WalKR in controlling S. aureus capsule production. Third, whether blocking WalKR-MgrA affects S. aureus virulence is not determined. The in vivo experiments are important for the development of alternative anti-virulence factor therapies, and we will perform these experiments in future.

Author Contributions

Conceptualization, Z.X. and X.R.; methodology, Z.G., H.P., W.S. and Y.Y.; investigation, Z.G., H.P., Y.R., X.H. and J.D.; resources, Z.H.; data curation, Z.G., H.P. and Z.H.; writing—original draft, Z.G. and H.P.; writing—review and editing, Z.X. and X.R.; visualization, Y.R.; supervision, X.R.; project administration, Z.X. and X.R.; funding acquisition, X.R. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Investigations only involved mouse blood that was purchased from SCBS, China. Therefore, no specific ethical approval was required.

Informed Consent Statement

Not applicable.

Data Availability Statement

The RNA-seq data in this study are deposited in the GEO DataSets under the ID code of GSE127706.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1. WalK(S221P) mutation promotes the capsule production of S. aureus. (A) The heat map showing the transcriptomic analysis of 16 cap genes in S. aureus XN108 versus XN108-R. 1, 2, and 3 represent the three biologically repeated samples. (B) RT-qPCR detection of cap genes capA, capG, and capL in XN108 and XN108-R. The gyrB was used as a reference gene for normalization. The data are expressed as the mean ± standard derivation (SD, n = 3). (C) Quantitative analysis of capsular polysaccharides with the phenol–sulfuric acid method. The data are expressed as the mean ± SD (n = 3). (D) Capsule thickness comparison. Data were expressed as mean ± SD (XN108, n = 12; XN108-R, n = 20). (E) Representative TEM images of XN108 and XN108-R. The cell wall and capsule are indicated by white and yellow triangles, respectively. Statistical significance in (B–D) was calculated by Student’s t-test; * represents p [less than] 0.05, ** indicates p [less than] 0.01, and *** shows p [less than] 0.001.

Figure 2. WalK(S221P) mutation indirectly controls cap expression. (A) β-Galactosidase assay. The pOS-cap reporter plasmid was transformed into XN108 and XN108-R, and the LacZ activity was detected. The data are indicated as mean ± SD (n = 3). Statistical significance was calculated by Student’s t-test, * represents p [less than] 0.05. (B) SDS-PAGE analysis of the purified WalK and WalR. (C) EMSA showing the DNA-binding ability of WalK-activated WalR to the promoter DNA fragment of the cap gene (Pcap). The promoter DNA fragment of agr (Pagr) was used as a positive control.

Figure 3. WalK(S221P) mutation increases cap expression through MgrA. (A) RT-qPCR detection of the regulator genes in XN108 and XN108-R. The gyrB gene was used as a reference for normalization. The data are expressed as mean ± SD (n = 3). (B) β-Galactosidase assay. The pOS-mgrA reporter plasmid was transformed into XN108 and XN108-R, respectively, and the LacZ activity was detected. The data are presented as mean ± SD (n = 3). (C) The EMSA of WalK-activated WalR with the biotin-labelled mgrA promoter DNA probe (PmgrA). The unlabelled probe was used as the specific competitor, while the unlabelled partial fragment of hu ORF region was used as the non-specific competitor. (D) β-Galactosidase assay. The pOS-cap plasmid was transformed into XN108 and XN108ΔmgrA, respectively, and the LacZ activity was detected. Data are presented as mean ± SD (n = 3). (E) SDS-PAGE analysis of the purified MgrA proteins. (F) The EMSA of MgrA with the biotin-labelled cap gene promoter DNA probe (Pcap). Statistical significance in (A,B,D) was calculated by Student’s t-test; ns shows no significance, ** indicates p [less than] 0.01, and *** represents p [less than] 0.001.

Figure 4. Deletion of mgrA reduces capsule production in S. aureus. (A) Growth curves of XN108 and its derivatives. The data represent the mean ± SD of each time point (n = 3). (B) Representative TEM images of XN108 and its derivatives. The cell wall and capsule are indicated. (C) Capsular thickness of XN108 and its derivatives. (D) Capsular polysaccharide quantification with the phenol–sulfuric acid method. Statistical significance was calculated by Student’s t-test; * indicates p [less than] 0.05 and *** represents p [less than] 0.001.

Figure 5. WalK(S221P) mutation and MgrA promotes the intracellular survival of S. aureus. (A) Whole blood killing. S. aureus XN108 and its derivatives were cultured in the BHI broth overnight. On the next day, bacterial cells were collected and co-cultured with mouse whole blood for 1 or 2 h at 37 °C. The survival rate of S. aureus cells was calculated after bacterial counting. (B) Bacterial count of XN108 and its derivatives 4 and 24 h postphagocytosis by RAW264.7 macrophages. The data represent the mean ± SD (n = 3). The statistical significance was calculated by two-way ANOVA. * p [less than] 0.05, ** p [less than] 0.01, *** p [less than] 0.001, and ns indicates no significance.

Figure 6. The schematic diagram showing the role of the WalK(S221P) mutation in promoting S. aureus capsule production through an MgrA-dependent pathway. The WalK(S221P) mutation in the 3D schematic models was indicated by red balls.

Table 1

Strains and plasmids used in this study.

Strains/Plasmids	Characteristics	Source/Reference
strains
S. aureus strain
XN108	A vancomycin-intermediate and methicillin-resistant strain with WalK(S221P) mutation carrying CP8 capsule	[26]
XN108-R	XN108 derivative with WalK(S221P) mutation recovered	[26]
Newman	MSSA strain carrying CP5 without WalK(S221P) mutation	[26]
K-Newman	Newman derivative with WalK(S221P) mutation introduced	[26]
XN108ΔmgrA	XN108 derivative with seamless deletion of mgrA gene	This work
XN108ΔmgrA/pLI-mgrA	XN108ΔmgrA complemented with the mgrA gene	This work
XN108-pOS-mgrA	XN108 derivative with plasmid pOS-mgrA	This work
XN108-R-pOS-mgrA	XN108-R derivative with plasmid pOS-mgrA	This work
XN108-pOS-cap	XN108 derivative with plasmid pOS-cap	This work
XN108ΔmgrA-pOS-cap	XN108ΔmgrA derivative with plasmid pOS-cap	This work
E. coli strain
DH5α	Cloning host for maintaining recombinant plasmids	Biomed
BL21(DE3)	Expression host for exogenous protein production	Biomed
plasmids
pBT2	Shuttle vector, temperature sensitive, AmpR, and CmR	[31]
pLI50	Expression vector, AmpR, and CmR	[31]
pOS1	Reporter vector with lacZ coding sequence for the β-galactosidase assay, CmR	[31]
pET28a	Expression vector, KanR	[31]

Cm^R, chloramphenicol resistant; Kan^R, kanamycin resistant; Amp^R, ampicillin resistant.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13030502/s1, Figure S1: WalK(S221P) mutation promotes capsule production of S. aureus strain Newman; Figure S2: Characterization of the mgrA deletion mutant XN108ΔmgrA; Table S1: Primers used in this study.

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